Incorporation of radioactive amino acids into protein in isolated rat hepatocytes

The incorporation of radioactivity from a 14C-labelled amino acid mixture (algal protein hydrolysate) into protein in isolated rat hepatocytes has been studied. The incorporation rate declined with increasing cell concentration, an effect which could be explained partly by isotope consumption, partly (and largely) by isotope dilution due to the formation of non-labelled amino acids by the cells. At a high extracellular amino acid concentration, the rate of incorporation into protein became independent of cell concentration, because the isotope dilution effect was now quantitatively insignificant. The time course of protein labelling at various cells concentrations correlated better with the intracellular than with the extracellular amino acid specific activity, suggesting that amino acids for protein synthesis were taken from an intracellular pool. With increasing extracellular amino acid concentrations both the intracellular amino acid concentration, the intracellular radioactivity and the rate of incorporation into protein increased. Protein labelling exhibited a distinct time lag at high amino acid concentrations, presumably reflecting the time-dependent expansion of the intracellular amino acid pool. The gradual increase in the rate of protein labelling could be due either to an increased intracellular specific activity, or to a real stimulation of protein synthesis by amino acids, depending on whether the total intracellular amino acid pool or just the expandable compartment is the precursor pool for protein synthesis.     

The effect of angiotensin II on platelet intracellular free magnesium and calcium ionic concentrations in essential hypertension

OBJECTIVE: To assess the effects of angiotensin II on intracellular free Mg2+ and Ca2+ concentrations in platelets from normotensive and hypertensive subjects. DESIGN AND METHODS: Seventeen normotensive, 25 untreated hypertensive and 18 treated hypertensive patients were studied. Intracellular Mg2+ concentrations were measured with the fluorescent dye mag-fura-2-acetyoxymethylester (AM) and intracellular Ca2+ concentrations with the fluorescent dye fura-2AM under basal conditions and after stimulation by angiotensin II, saralasin (angiotensin II antagonist), arginine vasopressin and endothelin-1. The effects of increased extracellular Mg2+ concentrations on intracellular Mg2+ and Ca2+ concentrations were also determined. RESULTS: The intracellular basal Ca2+ concentration was significantly higher in the untreated hypertensives compared with the normotensives and treated hypertensive subjects (150 +/- 14 nmol/l versus 120 +/- 17 nmol/l for normotensives and 124 +/- 8 nmol/l for treated hypertensives). The basal intracellular Mg2+ concentration was significantly lower in the untreated hypertensive compared to the normotensive and treated hypertensive groups (0.37 +/- 0.08 mumol/l versus 0.58 +/- 0.09 mumol/l for normotensives and 0.52 +/- 0.11 mumol/l for treated hypertensives). In the hypertensive groups, inverse correlations were found between intracellular Ca2+ and intracellular Mg2+ concentrations (r = -0.44, P < 0.05) and between intracellular Mg2+ and diastolic blood pressure (r = -0.35, P < 0.05), while a positive correlation was found between intracellular Ca2+ and systolic blood pressure (r = 0.41, P < 0.05). Exposure of the platelets to 1 nmol/l angiotensin II significantly increased intracellular Ca2+ and significantly decreased intracellular Mg2+ concentrations in all three groups. The angiotensin II-evoked effect on intracellular Ca2+ was exaggerated in the untreated hypertensives and blunted in the treated patients (basal versus stimulated: 150 +/- 14 versus 217 +/- 20 nmol/l in untreated hypertensives; 124 +/- 8 versus 140 +/- 10 nmol/l in treated hypertensives). Saralasin (0.1 mumol/l) abolished the effects of angiotensin. Arginine vasopressin (1 mumol/l) increased the intracellular Ca2+ concentration, whereas endothelin-1 (1 nmol/l) had no significant effect on either intracellular Ca2+ or intracellular Mg2+. Increasing extracellular Mg2+ concentrations led to significant reductions in intracellular Ca2+ concentrations in all groups and a significant elevation of the intracellular Mg2+ concentration in the untreated hypertensive patients only. CONCLUSIONS: These data demonstrate a relationship between angiotensin II and intracellular magnesium and calcium. In hypertension, angiotensin II-stimulated calcium responses may be related to simultaneously decreased intracellular magnesium concentrations.     

Regulation of fecal bile acid excretion in male golden Syrian hamsters fed a cereal-based diet with and without added cholesterol

The objective of these studies was to investigate the comparative physiology and regulation of bile acid metabolism in the male Golden Syrian hamster by measuring the rate of fecal bile acid excretion and bile acid pool size in animals fed a cereal-based diet either alone, or with added cholesterol or cholestyramine. In group-housed hamsters fed only the plain diet fecal bile acid excretion in animals at 6, 10, and 15 weeks of age averaged 11.0, 8.0, and 6.9 mumol/d per 100 g body weight (bw), respectively. Pool size, measured by subtracting from the total amount of bile acid washed out over 12 hours of biliary diversion the amount of bile acid excreted in the stools over the same period, equalled 17.8 mumol/100 g bw in 15-week-old hamsters fed the plain diet. Hence, under basal conditions, these animals turned over about 38% of their bile acid pool daily. In hamsters fed a diet with 3% cholestyramine for 18 days, fecal bile acid excretion averaged 20.6 mumol/d per 100 g bw, and the pool size contracted to 5.8 mumol/100 g bw. In matching animals fed a diet containing 0.12% cholesterol for 30 days, hepatic cholesterol levels increased from 1.9 +/- 0.1 to 12.6 +/- 0.7 mg/g, fecal bile acid excretion increased marginally from 5.8 to 8.0 mumol/day per 100 g bw, while pool size was unchanged (16.6 mumol/100 g bw). When the cholesterol content of the diet was raised to 1.0%, hepatic cholesterol levels reached 66.5 +/- 2.6 mg/g, but bile acid excretion remained at 8 mumol/d per 100 g bw. These data define some of the basal features of bile acid metabolism in the hamster, and substantiate the view that the marked cholesterolemic response of this species may relate partly to a limited ability to convert dietary cholesterol to bile acid.     

Vasopressin reduces taurochenodeoxycholate-induced hepatotoxicity by lowering the hepatocyte taurochenodeoxycholate content

BACKGROUND/AIMS: Vasopressin has been reported to reduce bile flow, but its effects on bile acid secretion and bile acid-related hepatotoxicity are still unclear. We therefore investigated the influence of vasopressin on the hepatotoxicity and biliary excretion of taurochenodeoxycholic acid in primary cultured rat hepatocytes and isolated perfused rat liver models. METHODS/RESULTS: 1) Addition of vasopressin to hepatocyte cultures significantly decreased lactate dehydrogenase release as compared to cultures exposed to 1 mM taurochenodeoxycholic acid alone, and also reduced intracellular taurochenodeoxycholic acid content from 19.3 +/- 2.2 to 13.0 +/- 1.6 nmol/mg protein. After 30 min of preincubation with 1 mM taurochenodeoxycholic acid, rinsing and reculture of hepatocytes in bile acid-free medium resulted in gradual decrease in the intracellular level of the bile acid, and addition of vasopressin (10(-9) M) to the reculture medium accelerated this process. 2) Superimposition of vasopressin (330 pmol/l) for 10 min on taurochenodeoxycholic acid infusion (1.0 mumol/min: 25 mumol/l) caused a rapid increase in bile flow and biliary excretion of taurochenodeoxycholic acid (697 +/- 42 vs 584 +/- 27 nmol/10 min per g liver) from perfused rat livers, and significantly reduced lactate dehydrogenase release. 3) Superimposition of the PKC blocker H-7 (5 mumol/l) on taurochenodeoxycholic acid infusion (1.0 mumol/min: 25 mumol/l) caused a gradual increase in bile flow and biliary excretion of taurochenodeoxycholic acid. Furthermore, an additional infusion of vasopressin (100 pmol/l) for 10 min in the presence of H-7 produced a greater increase in bile flow and biliary excretion of taurochenodeoxycholic acid as compared with H-7 alone (754 +/- 71 vs. 657 +/- 26 nmol/g liver). 4) Continuous infusion of vasopressin (330 pmol/l) significantly increased the late peak (10-50 min) of horseradish peroxidase excretion from perfused livers (from 8.48 +/- 1.02 to 21.7 +/- 6.02 ng/g liver). CONCLUSIONS: These findings suggest that vasopressin exerts a protective effect against taurochenodeoxycholic acid-induced hepatotoxicity by stimulating the secretion of this bile acid via intracellular vesicular transport systems.     

Homeobox genes in hematopoiesis and leukemogenesis

The effect of cold and warm intermittent antegrade blood cardioplegia, on the intracellular concentration of taurine in the ischaemic/reperfused heart of patients undergoing aortic valve surgery, was investigated. Intracellular taurine was measured in ventricular biopsies taken before institution of cardiopulmonary bypass, at the end of 30 min of ischaemic arrest and 20 min after reperfusion. There was no significant change in the intracellular concentration of taurine in ventricular biopsies taken after the period of myocardial ischaemia in the two groups of patients (from 10.1 +/- 1.0 to 9.6 +/- 0.9 mumol/g wet weight for cold and from 9.3 +/- 1.3 to 10.0 +/- 1.3 mumol/g wet weight for warm cardioplegia, respectively). Upon reperfusion however, there was a fall in taurine in both groups but was only significant (P < 0.05) in the group receiving cold blood cardioplegia (6.9 +/- 0.8 mumol/g wet weight after cold blood cardioplegia versus 8.0 +/- 0.8 mumol/g wet weight following warm blood cardioplegia). Like taurine, there were no significant changes in the intracellular concentration of ATP after ischaemia in the two groups of patients (from 3.2 +/- 0.32 to 2.95 +/- 0.43 mumol/g wet weight for cold and from 2.75 +/- 0.17 to 2.62 +/- 0.21 mumol/g wet weight for warm cardioplegia, respectively). However upon reperfusion there was a significant fall in ATP in both groups with the extent of the fall being less in the group receiving warm cardioplegia (1.79 +/- 0.19 mumol/g wet weight for cold and 1.98 +/- 0.27 mumol/g wet weight for warm cardioplegia, respectively). This work shows that reperfusion following ischaemic arrest with warm cardioplegia reduces the fall in tissue taurine seen after arrest with cold cardioplegia. Accumulation of intracellular sodium provoked by hypothermia and a fall in ATP, may be responsible for the fall in taurine by way of activating the sodium/taurine symport to efflux taurine.     

No net splanchnic release of glutathione in man during N-acetylcysteine infusion

Glutathione and amino acid concentrations were measured in arterial and hepatic vein plasma in four healthy volunteers and two patients with cirrhosis. There was no significant splanchnic efflux of glutathione (95% confidence limits, -0.501 to 0.405 mumol/min). After infusion of N-acetylcysteine (NAC) in a high dose (150 mg/kg body weight primer plus 15 mg/(h x kg BW), corresponding to treatment of acetaminophen overdose, there was no change in the splanchnic glutathione efflux (95% confidence limits, -0.531 to 0.375 mumol/min). NAC increased hepatic plasma flow rate from 0.90 +/- 0.531 min-1 to 0.97 +/- 0.11 (mean +/- SEM; p < 0.05). The effects of NAC treatment on plasma amino acids corresponded to an increased load on hepatic metabolic N conversion and transamination among nonessential amino acids. Splanchnic uptake of serine, alanine, cystine, isoleucine, and phenylalanine increased after NAC compatible with stimulated hepatic glutathione synthesis. In contrast to the rat, plasma glutathione in man probably originates mainly from extrahepatic tissues.     

The influence of oxygen deficiency and electrical stimulation on the concentrations of ATP, ADP, AMP and phosphotaurocyamine in the body-wall musculature of Arenicola marina

1) Concentrations of ATP, ADP, and AMP, as well as phosphotaurocyamine in the body-wall musculature of Arenicola marina were determined enzymatically and by means of isotachophoresis after 12, 24, and 48 h of experimental anaerobiosis and after electrical stimulation under aerobic and anaerobic conditions. 2) Freshly collected lugworms contained 3.5-3.8 mumol ATP, 0.8-1.0 mumol ADP, and 0.3-0.5 mumol AMP, as well as 4.5-4.7 mumol phosphotaurocyamine per g wet weight (energy charge 0.81-0.85). 3) After 48 h of anaerobiosis the concentration of ATP was decreased to one half, that of phosphotaurocyamine to one third of the initial value, while the content of ADP on the other hand nearly doubled and that of AMP trebled (energy charge 0.60-0.63). 4) Electrical stimulation in the absence of oxygen caused a rapid decrease of the ATP and phosphotaurocyamine to low levels corresponding to those found after long-term anaerobiosis; the ADP and AMP concentrations were slightly higher than those found after long-term anaerobiosis (energy charge 0.55-0.58).     

Intermittent ischemia: energy metabolism, cellular volume regulation, adenosine and insights into preconditioning

Interruption of ischemia by brief reperfusions (I/R) is better tolerated by the heart than continuous ischemia. The present study aims to determine the metabolic profiles of isolated rat hearts during intermittent ischemia, the possible cardioprotective role of adenosine and the influence of I/R on intracellular volumes, using multinuclear NMR spectroscopy. After five I/R (5/5 min) episodes, hearts paced at 5 Hz developed pressures comparable to those of hearts continuously perfused for 50 min at 37 degrees C (CP). Following the first 5 min episode of no-flow ischemia, [ADP] dropped from 72 +/- 9 to 43 +/- 5 microM (P < 0.001) and remained stable at the end of the following reperfusions, despite a 2.5-4-fold increase during each episode of 5 min ischemia. Intracellular volumes were stable during CP at a value of 2.50 +/- 0.06 ml/g dry weight, and decreased by 4, 8, and 12% after 1, 3, and 5 I/R episodes. The phosphorylation potentials decreased from 54 +/- 8 to 4 mM-1 during each period of 5 min ischemia and were 40 +/- 6 and 28 +/- 6 mM-1 after CP and I/R5, respectively. Cardiac glycogen had decreased during 50 min of CP from 103 +/- 13 to 81 +/- 9 mumol/g dry weight and lactate production was 116 +/- 15 mumol/heart. Five I/R episodes decreased glycogen to 46 +/- 7 mumol/g dry weight (P < 0.005 v CP) and increased lactate efflux to 262 +/- 31 mumol/ heart (P < 0.005 v CP). These findings suggest that a brief ischemia/reperfusion episode increases anaerobic metabolism of exogenous glucose, reduces [ADP] and induces cellular shrinkage. Administration of the adenosine receptor blocker 8-phenyl theophylline (8PT) during intermittent perfusion depressed the developed pressure to 78 +/- 7%, accentuated the decrease in phosphorylation potential (14 +/- 4 mM-1), abolished cellular shrinkage, reduced lactate efflux and blunted the decrease in ADP following the first I/R episode. In variance, no detectable changes were observed during intermittent ischemia when the ATP-sensitive potassium channel blocker glibenclamide was administered. These data demonstrate: (a) a brief episode of ischemia/reperfusion stimulates anaerobic metabolism of exogenous glucose and lowers intracellular ADP concentration: (b) adenosine receptors are partially responsible for the glycolytic stimulation during intermittent ischemia; (c) cellular shrinkage is related to the rate of glycolysis during intermittent ischemia/reperfusion.     

Effect of spinal kainic acid receptor activation on spinal amino acid and prostaglandin E2 release in rat

Current work has shown that spinal excitatory amino acid receptor activation can evoke physiological phenomena that may be mediated by the subsequent depolarization of glutamate-containing neurons and the activation of cyclo-oxygenase systems. To investigate this phenomenon, rats were implanted with lumbar intrathecal loop dialysis catheters for perfusion and an additional lumbar intrathecal PE-10 catheter for drug delivery. Two days after implantation, kainic acid (1 microgram) was injected intrathecally under light (0.5%) halothane anaesthesia and the spinal release of several amino acids and prostaglandin E2 was examined. Resting concentrations (mean expressed as pmol/25 microliters) of glutamate (89), aspartate (9), serine (387), glycine (597), taurine (185), asparagine (113) and prostaglandin E2 (0.43) were observed. Intrathecal kainic acid produced significant signs of arousal in the rat and evoked a significant increase (mean +/- S.E.M. of % baseline concentration) in aspartate (445 +/- 127%) and glutamate (221 +/- 35%). Prostaglandin E2 concentration was increased in the second post-injection sample (180 +/- 36%). Intrathecal pretreatment with 6-cyano-7-nitroquinoxaline-2, 3-dione (3 micrograms or 10 micrograms), a non-N-methyl-D-aspartate receptor antagonist, blocked amino acid but not prostaglandin E2 release after kainic acid injection. Pretreatment with MK-801 (10 micrograms; non-competitive NMDA receptor antagonist) had no significant effect on evoked release of amino acids or prostaglandin E2. Indomethacin (10 micrograms, a cyclo-oxygenase inhibitor) pretreatment significantly decreased baseline prostaglandin E2 release in control animals (61 +/- 6%) and suppressed kainic acid-evoked aspartate, taurine and prostaglandin E2 release, but had no effect on the concentration of glutamate after kainic acid injection. These data suggest that activation of spinal kainic acid receptors provides a powerful stimulus for secondary excitatory amino acid release and, consistent with the concurrent appearance of prostaglandin E2, that this release is potentiated by the release of a cyclo-oxygenase product.     

Exogenous insulin reduces proteolysis and protein synthesis in extremely low birth weight infants

OBJECTIVE: To determine the effect of a continuous insulin infusion on protein and glucose metabolism in extremely low birth weight (ELBW) infants. STUDY DESIGN: We measured the rate of appearance (Ra) of the essential amino acids leucine and phenylalanine (reflecting proteolysis), utilization of phenylalanine for protein synthesis, and glucose Ra using stable isotope tracers during a basal infusion of glucose (6 mg/kg/min) and in response to a continuous infusion of insulin (0.05 U/kg/hr) by means of the euglycemic hyperinsulinemic clamp technique. Four clinically stable, euglycemic ELBW infants (26 +/- 0 weeks' gestation, 894 +/- 44 gm birth weight, 2.8 +/- 0.8 days of age) were studied. RESULTS: In response to a greater than tenfold increase in insulin concentration (from 7 +/- 2 to 79 +/- 13 microU/ml), there was a 20% decrease in leucine Ra (Basal: 272 +/- 27 mumol/kg/hr; Insulin: 226 +/- 29 mumol/kg/hr; p < 0.01) and in phenylalanine Ra (Basal: 91 +/- 5 mumol/kg/hr; Insulin: 72 +/- 2 mumol/kg/hr; p < 0.05). Use of phenylalanine for protein synthesis also decreased by a similar magnitude (Basal: 77 +/- 4 mumol/kg/hr; Insulin: 62 +/- 1 mumol/kg/hr; p < 0.05). Glucose utilization doubled (from 8 +/- 0.9 to 15.7 +/- 1.1 mg/kg/min; p = 0.0003) and plasma lactate concentrations tripled (from 2.1 +/- 0.5 to 5.7 +/- 1.0 mmol/L; p < 0.05) during the insulin infusion. CONCLUSIONS: During an infusion of glucose alone, pharmacologic concentrations of insulin in ELBW infants produced no net protein anabolic effect. Furthermore, euglycemic hyperinsulinemia was accompanied by development of significant metabolic acidosis.     

Interactions between excitatory amino acids and tachykinins in the rat spinal dorsal horn

Whole-cell patch-clamp technique of freshly isolated rat spinal dorsal horn (DH) neurons, intracellular recording from DH neurons in a slice preparation, and high performance liquid chromatography with fluorimetric detection of release of endogenous glutamate and aspartate from spinal cord slice following activation of primary afferent fibers were employed to investigate interactions between excitatory amino acids (EAA) and tachykinins [substance P (SP) and neurokinin A (NKA)]. Potentiation of N-methyl-D-aspartate (NMDA)-, quisqualate (QA)- and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-, but not kainate-induced currents by SP and NKA was found. Spantide II, a claimed novel nonselective tachykinin antagonist, effectively blocked the SP (2 nM)-induced potentiation of the responses of DH neurons to NMDA. In the presence of glycine (0.1 microM), the SP-evoked increase of the NMDA-induced current was prevented. However, 7-chlorokynurenic acid (2 microM), a competitive antagonist at the glycine allosteric site of the NMDA receptor, led to the reestablishment of the SP effect. Brief high frequency electrical stimulation of primary afferent fibers produced a long-lasting potentiation of presumed monosynaptic and polysynaptic excitatory postsynaptic potentials and sustained enhanced release of endogenous glutamate (218.3 +/- 66.1%) and aspartate (286.3 +/- 58.0%). Possible functional implications of the observed phenomena are discussed in relation to transmission and integration of sensory information, including pain.     

The mechanism of potentiation of the antitumor effect of 5-fluorouracil by methionine-free intravenous amino acid solution (AO-90) in rats

AO-90, a methionine-free intravenous amino acid solution (7.43%) showed to potentiate the antitumor effect of 5-fluorouracil (5-FU) when concomitantly used as the nitrogen source in total parenteral nutrition (TPN) in Yoshida sarcoma (YS)-bearing rats. In the present experiment, this potentiation mechanism was studied by determining the serum methionine level and tumor methylenetetrahydrofolate (CH2FH4) content in YS-bearing Donryu rats given AO-90 (nitrogen 0.73g/kg on the 1st day and 1.46g/kg for the remaining 6 days) by TPN for 1 week. The rats were subcutaneously inoculated with 10(4) YS cells in the dorsum 3 days before the start of TPN. Inhibition of thymidylate synthase activity in tumor tissue after dosing of AO-90 (nitrogen 0.68g/kg on the 1st day and 1.36 g/kg for the remaining 6 days) by TPN along with daily intraperitoneal dosing of 5-FU (10 mg/kg) was also evaluated with the inoculation of 10(6) tumor cells. The results were compared with those in tumor-bearing rats given TPN with a commercially available amino acid solution containing methionine. On day 5 of TPN, the tumor-bearing rats given AO-90 showed a significantly lower serum methionine level than the control rats: 101 +/- 11 mumol/l versus 29 +/- 14 mumol/l (p < 0.01); and a higher CH2FH4 content in tumor: 7.0 +/- 2.8 pmol/g protein versus 23.7 +/- 16.6 pmol/g protein (p < 0.05). Thymidylate synthase inhibition was 81.2 +/- 5.1% in the AO-90 group and 30.9 +/- 26.3% in the control group (p < 0.01). The results of the present study suggest that AO-90 potentiate the antitumor effect of 5-FU by biochemical modulation. AO-90 concomitantly given with 5-FU for 7 days was effective not only in the allogeneic tumor model, but also in WKAH and SHR rats previously inoculated with 10(6) of syngeneic KDH-8 hepatoma cells and SST-2 adenocarcinoma cells, respectively. Weight of SST-2 adenocarcinoma in SHR rats after the TPN period was significantly smaller in the AO-90 group than in the control rats given methionine-containing TPN and 5-FU: 2.66 +/- 0.91 versus 5.12 +/- 2.11 (p < 0.05).     

Activity of some serum enzymes in calves suffering from white muscle disease

The paper described the findings of the activity of aspartate amino transferase (GOT) and alanine amino transferase (GPT), lactate dehydrogenase (LDH), alkaline phosphatase (AP), and aldolase in the blood serum of calves examined for white-muscle disease (WMD). Relapsing mass accurrence of the disease was reported from various agricultural enterprises where calves were fed a milk replacer without vitamin E. In comparison with clinically healthy calves fed a feed mixture with vitamin E, calves suffering from the clinical form of WMD showed an alkaline phosphatase level decrease from 32.3 +/- 7.6 u. K. A. to 15.1 +/- 8.2 u. K. A. On the other hand, the activites of ALD, GOT, GPT, and LDH showed a statistically significant increase. The acute and sub-acute course of the disease increased enzyme activities as follows: ALD from 4.2 +/- 1.1 mumol (= 70.0 +/- 17.0 i.u.) to 9.7 +/- 2.1 mumol (= 163.0 +/- 33.2 i. u.), GOT from 0.9 +/-0.5 mumol (= 68.0 +/- 5.8 i.u.) to 16.7 +/- 11.7 mumol (= 567.0 +/-40.0 i. u.) GPT from 0.2 +/- 0.8 mumol (= 5.0 +/- 12.4 i. u.) to 9.8 +/- 2.8 mumol (= 330.0 +/- 40.4 i.u.), LDH from 46.1 +/- 5.4 mumol (= 765.0 +/- 40.0 i.u.) to 72.7 +/- 24.3 mumol (= 1,207.0 +/- 403.0 i.u.). In WMD-affected herds, similar enzyme activity fluctuations were observed even in calves showing no clinical signs of the disease. It follows from the study that the examination of serum enzymes provides a method to demonstrate the clinical and pre-clinical forms of white-muscle disease and that it can be included in the set of tests for the diagnosis of diseases in calves. The significant differences in all calves in the affected herds show that the disease is a danger to all animals in the herd fed a deficient mixture.     

Hepatic release of erythropoietin induced by transarterial chemoembolization in patients with hepatocellular carcinoma

It has been shown previously that erythropoietin expression in vitro by hepatoma cells increases in response to hypoxia. To verify whether hypoxia of the tumor might result in hepatic release of erythropoietin in vivo, serum erythropoietin concentrations were measured immunoenzymatically in 12 patients (5 women, 7 men) who underwent transarterial chemoembolization for hepatocellular carcinoma. Peripheral blood samples were collected at baseline, and after 6 hours and 1, 2, 3, and 7 days after the procedure. In a second set of experiments, performed in three male patients also undergoing chemoembolization for hepatocellular carcinoma, paired blood samples were collected after catheterization of the hepatic veins and of the right antecubital vein. None of the patients had erythrocytosis. In comparison with a baseline mean value +/- SEM of 100.6 +/- 12.6 micrograms/L, serum erythropoietin concentrations were the following; +6 hours, 55.4 +/- 18.0 (P < .001); +1 day, 102.4 +/- 24.7 (P = NS), +2 days, 183.0 +/- 31.1 (P < .05); +3 days, 155.0 +/- 26.0 (P < .05); +7 days, 153.3 +/- 27.4 (P < .05) (matched Student's t-test). The ratio of hepatic vein/antecubital vein serum erythropoietin concentrations increased from 0.85 at baseline to 1.30 at +2 days, paralleling the increase of aspartate transaminase (r = .914, P < .005). After chemoembolization, no correlation was found between serum erythropoietin and alpha-1-fetoprotein concentrations. The concentration of the latter, stable initially, decreased 7 days after the procedure.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lipoic acid reduces glycemia and increases muscle GLUT4 content in streptozotocin-diabetic rats

Alpha lipoic acid (lipoate [LA]), a cofactor of alpha-ketodehydrogenase, exhibits unique antioxidant properties. Recent studies suggest a direct effect of LA on glucose metabolism in both human and experimental diabetes. This study examines the possibility that LA positively affects glucose homeostasis in streptozotocin (STZ)-induced diabetic rats by altering skeletal muscle glucose utilization. Blood glucose concentration in STZ-diabetic rats following 10 days of intraperitoneal (i.p.) injection of LA 30 mg/kg was reduced compared with that in vehicle-treated diabetic rats (495 +/- 131 v 641 +/- 125 mg/dL in fed state, P = .003, and 189 +/- 48 v 341 +/- 36 mg/dL after 12-hour fast, P = .001). No effect of LA on plasma insulin was observed. Gastrocnemius muscle crude membrane GLUT4 protein was elevated both in control and in diabetic rats treated with LA by 1.5- and 2.8-fold, respectively, without significant changes in GLUT4 mRNA levels. Gastrocnemius lactic acid was increased in diabetic rats (19.9 +/- 5.5 v 10.4 +/- 2.8 mumol/g muscle, P < .05 v nondiabetic rats), and was normal in LA-treated diabetic rats (9.1 +/- 5.0 mumol/g muscle). Insulin-stimulated 2-deoxyglucose (2 DG) uptake into isolated soleus muscle was reduced in diabetic rats compared with the control group (474 +/- 15 v 568 +/- 52 pmol/mg muscle 30 min, respectively, P = .05). LA treatment prevented this reduction, resulting in insulin-stimulated glucose uptake comparable to that of nondiabetic animals. These results suggest that daily LA treatment may reduce blood glucose concentrations in STZ-diabetic rats by enhancing muscle GLUT4 protein content and by increasing muscle glucose utilization.     

The effects of various diphosphonates on a rat model of cardiac calciphylaxis

Compartmental analysis of the disappearance curve of serum cholesterol specific activity after an intravenous administration of a tracer does of cholesterol-4-14C was carried out in five patients with resection of the distal small intestine, cecum, and proximal colon. The data fit best a two compartment model in all five cases with the rapidly exchangeable pool of 16.6+/-3.2 g (mean +/- SD, 60% of the mean of 15 normal subjects) and the slowly exchangeable pool of 31.5 +/- 10.9 g (65%). The reduction of the pool sizes was associated with a shorter mean transit time of cholesterol, 22.15 +/- 8.07 days (40%) and increased turnover rate, 2.42 +/- 0.72g/ day (172%). Direct fecal analysis for the neutral sterols and bile acids derived from the exchangeable pool confirmed the turnover rate obtained from the compartmental analysis. The increased fecal excretion was mainly in the bile acid fraction. The study suggests that the ileal and proximal colon resection results in bile acid malabsorption which, in turn, increases hepatic cholesterol and bile acid synthesis. The synthetic rates, however, could not compensate totally for the excretory rate. Therefore, the pool size decreased to a new low steady state of equilibrium.     

Endothelium-dependent effect of perindopril and enalaprilat in rat thoracic aorta

AIM: To study the effect of the angiotensin-converting enzyme (ACE) inhibitors perindopril (Per) and enalaprilat (Ena) on the reactivity of the endothelium in normal rats. METHODS: Male rats were treated intragastrically with Per (2 mg.kg-1.d-1) or placebo (n = 18) for 6 wk. Aorta was isolated for experiment. Another set of isolated aortic rings with and without endothelium were incubated with Ena (0.1 mumol.L-1) for 30 min. Responses to acetylcholine, serotonin, phenylephrine, sodium nitroprusside (SN), and nitroglycerin (Nit) were observed. RESULTS: Endothelium-dependent relaxation to acetylcholine was augmented in aortic rings from rats treated with Per in comparison with control. The IC50 value (95% confidence limits) decreased from 3.8 (0.56-26.1) mumol.L-1 (control group) to 0.98 (0.28-3.41) mumol.L-1 (Per-treated group). The maximal relaxation was augmented from 62 +/- 9% to 78 +/- 10% (P < 0.01). However, the responses to the endothelium-independent vasodilators, SN and Nit, were similar. Serotonin- and phenylephrine-induced contractions were decreased, which were influenced by basal release of endothelium-derived relaxing factor (EDRF). EC50 values was 6.1 (2.6-14.4) nmol.L-1 vs 8.3 (3.6-18.8) nmol.L-1 in comparison with control group and Per-treated group. The maximal contraction was decreased from 2.42 +/- 0.29 g (control group) to 1.96 +/- 0.25 g (treated group) (P < 0.01). Similar results were found in incubation with Ena. CONCLUSION: Ena and Per enhanced the basic release of EDRF from vascular endothelium.     

Hydroperoxide metabolism in rat liver. K+ channel activation, cell volume changes and eicosanoid formation

Addition of t-butylhydroperoxide (0.2 mM) to isolated perfused rat liver led to a net K+ release of 7.2 +/- 0.2 mumol/g within 8 min and a net K+ reuptake of 6.6 +/- 0.4 mumol/g following withdrawal of the hydroperoxide, in line with earlier findings by Sies et al. [Sies, H., Gerstenecker, C., Summer, K. H., Menzel, H. & Flohé, R. (1974) in Glutathione (Flohé, L., Ben?hr, C., Sies, H., Waller, H. D., eds) pp. 261-276, G. Thieme Publ. Stuttgart]. Net K+ release roughly paralleled the amount of GSSG released from the liver under the influence of the hydroperoxide. The t-butylhydroperoxide-induced K+ efflux was inhibited by approximately 70% in the presence of Ba2+ (1 mM), by 30% in Ca(2+)-free perfusions and was decreased by 50-60% when the intracellular Ca2+ stores were simultaneously depleted by repeated additions of phenylephrine. t-Butylhydroperoxide-induced K+ efflux was accompanied by a decrease of the intracellular water space by 58 +/- 14 microliter/g (n = 4), corresponding to a 10% cell shrinkage. The effect of t-butylhydroperoxide on cell volume was inhibited by 70-80% in the presence of Ba2+. In isolated rat hepatocytes treatment with t-butylhydroperoxide led to a slight hyperpolarization of the membrane at concentrations of 100 nM, but marked hyperpolarization occurred at t-butylhydroperoxide concentrations above 10 microM. t-Butylhydroperoxide (0.2 mM) transiently increased the portal-perfusion pressure by 3.3 +/- 0.6 cm H2O (n = 18), due to a slight stimulation of prostaglandin-D2 release under the influence of the hydroperoxide. In the presence of Ba2+ (1 mM), t-butylhydroperoxide increased the perfusion pressure by 12.7 +/- 1.2 cm H2O (n = 9) and produced an approximately tenfold increase of prostaglandin-D2 and thromboxane-B2 release. Under these conditions, glucose output from the liver rose from 0.9 +/- 0.03 to 2.9 +/- 0.7 mumol.g-1.min-1 (n = 4) with a time course roughly resembling that of portal-pressure increase and prostaglandin-D2 overflow. These effects were largely abolished in the presence of ibuprofen or the thromboxane-receptor-antagonist BM 13.177. The t-butylhydroperoxide effects on perfusion pressure, glucose and eicosanoid output were also enhanced in the presence of insulin or during hypotonic exposure; i.e. conditions known to swell hepatocytes, but not during hyperosmotic exposure. The data suggest that t-butylhydroperoxide induces liver-cell shrinkage and hyperpolarization of the plasma membrane due to activation of Ba(2+)-sensitive K+ channels.(ABSTRACT TRUNCATED AT 400 WORDS)     

Balloon catheter systems for PTCA: the importance of the catheter length

This complex study was designed to measure the transport and excretion characteristics of gadolinium ethoxybenzyl diethylenetriaminepentacetic acid (Gd-EOB-DTPA) in dog's livers following bolus and infusion. Simultaneous T1 magnetic resonance imaging was performed to measure maximum signal enhancement. Anaesthetized dogs had cannulation of the common bile duct and urinary bladder for collections and cannulation of the femoral artery and vein for monitoring, blood sampling and infusion. Gd-EOB-DTPA was administered by bolus (range 12.5-200 mumol/kg) and infusion (range 0.4-6.4 mumol/min per kg). An hepatic transport maximum 0.09-0.15 mumol/min/kg was achieved with a blood concentration of 0.03-0.06 mumol/mL. Marked hepatic affinity for Gd-EOB-DTPA was demonstrated with measurements of liver concentration. Maximum T1 signal enhancement was achieved with blood Gd-EOB-DTPA concentration of 0.02-0.03 mumol/mL and a liver concentration of 1-2 mumol/g. The transport maximum for Gd-EOB-DTPA in the dog was similar to that for ipodate and iodipamide and effective imaging was achieved with sub-maximal doses. The maximum signal enhancement at blood concentrations less than required for maximum transport suggest a wide latitude for effective clinical imaging.     

Characterization of leucine transport by toadfish liver in vivo

Kinetic analysis of L-leucine uptake by toadfish liver at 20 degrees C in vivo has been carried out after pulse injection of L-[14C]leucine into the hepatic portal vein. D-[3H]mannitol, which is taken up slowly by toadfish liver, is used as a marker for extracellular space and space accessible by simple diffusion. At normal plasma leucine concentration (0.1 mM), leucine uptake occurs rapidly (t1/2 = 0.25 min), representing a flux of 0.6 mumol/min for the liver as a whole. Analysis of the distribution of radioactive leucine among intracellular and extracellular free pools and protein-bound form at times of 30 s to 5 min after injection is consistent with operation of a concentrative or uphill transport system accounting for 40% of uptake at normal plasma concentration. Saturation of uptake occurs at increasing leucine loads; calculation of intracellular pool dilution from protein synthesis data indicates that 20-30% of liver intracellular space is occupied by incoming leucine during the first 2 min after portal injection. Maximal flux (V max) is 4.1 mumol/min per 7-g liver as a whole with Km = 0.6 mM. Competitive inhibition of leucine uptake is afforded by isoleucine and phenylalanine with lesser effects by aspartic acid, cysteine, methionine, threonine, tyrosine, and valine. No effect is observed with alanine, glycine, histidine, lysine, and proline.